Introduction

C albicans is a dimorphic species capable of changing its morphology from the yeast form to the hyphal form, an all-important process to form mature biofilms and a transition critical to its pathogenesis1, 2, 3. Biofilm structure impairs the action of phagocytic cells from the immune system and antimicrobial agents4, 5, 6, which are notoriously difficult to eradicate as well as an important source of many recalcitrant infections.

Biofilm development occurs in three phases over a period of 24–48 h7, 8, 9. The initial phase begins with adherence of single cells to the substratum. Second, attached cells proliferate to form microcolonies and begin to deposit an extracellular matrix. Finally, once cells reach confluency, the network of yeast cells transits to filamentous (pseudohyphal and hyphal) forms, which become encased in the exopolymeric matrix. Baillie and Douglas10, 11 reported that slow growth of microorganisms, presumably due to some form of nutrient limitation, may contribute to mature biofilm resistance.

Beta-1,3 glucan is thought to be the main component of the three-dimensional matrix surrounding biofilm cells and closely tied to biofilm formation12. Nett et al13 showed that elevated beta-1,3 glucan levels were characteristic of biofilm cells as compared to planktonic free-living C albicans cells. A complex network of signaling pathways regulates yeast-hyphal morphogenesis. It is known that several signaling pathways, including Cph1-mediated mitogen-activated protein kinase and Efg1-mediated cyclic AMP/protein kinase A, participate in the regulation of morphological transition14, 15. These multiple pathways in conjunction with the pathway-specific transcription factors regulate the expression of hypha-specific genes, including ECE1, HWP1, HYR1, ALS1, ALS3, RBT1, RBT4, and HGC116, 17, 18, 19, 20, 21. Hyphal development is negatively regulated by transcriptional repressors Tup1, Nrg1, Sfl1, and Rfg122, 23, 24, 25. Sfl1 has a dual function in filamentous growth of C albicans: it acts as a repressor of hyphal development antagonizing activation of Flo8 but functions as an activator releasing from inhibition of Flo8 in the matrix at low temperature. Rfg1 is a second DNA-binding protein that targets Tup1 to promoters of hypha-specific genes. Tup1 functions with DNA binding proteins Nrg1 and Rfg1 as a transcription regulator to repress the expression of hypha-specific genes. Nrg1 is a zinc finger DNA-binding protein that represses hypha-specific genes in a Tup1-dependent manner, making a major contribution to the repression of hyphal development26.

Biomaterial infections are an increasingly alarming problem. Given their intrinsic recalcitrance to conventional therapies, new methods of examining these infections and a new class of antifungal drugs must be explored. 2-Amino-nonyl-6-methoxyl-tetralin muriate (10b), a 2-aminotetralin derivate, was synthesized as a novel chemical structural antifungal agent (Figure 1). In our previous study, we found that 10b had anti-biofilm activity. It has been demonstrated that farnesol was an extracellular quorum sensing molecule in the dimorphic fungus C albicans27, which inhibits the yeast to mycelium dimorphic transition and prevents biofilm formation28. Therefore, we selected farnesol as positive control to investigate the effect of 10b on C albicans biofilms formation. The aim of the work presented here was to further investigate the mechanism of action of 10b against C albicans biofilms at molecular level.

Figure 1
figure 1

Chemical structure of 2-amino-nonyl-6-methoxyl-tetralin muriate (10b).

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Materials and methods

Antifungal agents

Antifunal reagents used in the present experiment included farnesol (Alfa Aesar, A Johnson Matthey Co, Ward Hill, USA); miconazole (MCZ, Pfizer-Roerig Pharmaceuticals, New York, USA); 10b (Department of Medicinal Chemistry, School of Pharmacy, Second Military Medical University, Shanghai, China); and stock solutions of various concentrations in methanol (Sigma).

C albicans strains and growth conditions

C albicans SC5314 was used throughout this study. C albicans cells were propagated in yeast-peptone-dextrose (YPD) medium (1% w/v yeast extract, 2% w/v peptone, 2% w/v dextrose). Blastospores were harvested and washed twice in 0.15 mol/L phosphate-buffered saline (PBS; pH 7.2). The cells were then suspended in RPMI-1640 medium (RPMI 1640 supplemented with L-glutamine and buffered to pH 7.0 with morpholinepropanesulfonic acid (MOPS)) and counted in a haemocytometer and adjusted to 1.0×106 colony forming unit (CFU)/mL.

In vitro anti-biofilm activity assay

C albicans bioflm metabolic activity was assessed using the XTT reduction assay as described previously29. The principle is based upon the reduction of tetrazolium to tetrazolium formazan by mitochondrially active yeasts in the presence of menadione, an electron-coupling agent. Briefly, XTT (Sigma Chemical Co) at 0.5 mg/mL was added with menadione (10 mmol/L in acetone) to a final concentration of 1 μmol/L in PBS. A 200 μL aliquot of XTT-menadione was added to each well and incubated in the dark at 37 °C for 2 h. Any colorimetric change (a direct reflection of metabolic activity of the biofilms) was measured in a microtitre plate reader (Multiskan MK3) at 492 nm.

Biofilms were formed in selected 96-microtitre plates. Following the initial 1 h adhesion, the medium was aspirated and then washed thoroughly twice with sterile PBS before the addition of fresh RPMI-1640 medium containing different concentrations of antifungal agents and incubated for another 48 h at 37 °C. A series of antifungal-free wells was also included as controls. Sessile MIC50 were determined at 50% inhibition (SMIC50) compared with drug-free control wells using XTT reduction assay.

To determine the effect of 10b on adhesion of planktonic cells, antifungal drugs were added to a standardized suspension at zero time (preincubation) before it was added to a 96-microtiter plate. The plate was incubated for 90 min at 37 °C. Knowing that adherent populations on the substratum do not form a structurally differentiated community (biofilm) during this period of time30, we chose 90 min as the incubation time of the adhesion assay. To determine the effect of 10b on mature biofilms, mature biofilms (48 h) formed as above in the absence of antifungal drugs were added with fresh RPMI-1640 medium containing different concentrations of antifungal drugs. The plates were incubated at 37 °C for 3, 6, 12, and 24 h.

Confocal laser scanning microscopy

Confocal laser scanning microscopy (CLSM) was performed as described in the literature31 to demonstrate the inhibitory effect of 10b on biofilm formation. Formation of C albicans SC5314 biofilms was achieved by adding 4 mL standardized cell suspension to six-well plates containing plastic disks. Following 10b exposure and biofilm formation, the disks were removed and transferred to new six-well culture plates and incubated for 45 min at 37 °C in 4 mL PBS containing the fluorescent stains FUN-1 (10 μmol/L; Molecular Probes, Eugene, Oreg) and concanavalin A–Alexa Fluor 488 conjugate (ConA; 25 μg/mL; Molecular Probes). FUN-1 (excitation wavelength, 543 nm; emission wavelength, 560 nm; long-pass filter) is converted to an orange-red cylindrical intravacuolar structure by metabolically active cells, while ConA (excitation wavelength, 488 nm; emission wavelength, 505 nm; long-pass filter) binds to glucose and mannose residues of cell wall polysaccharides and emits a green fluorescence. After incubation with the dyes, the disks were flipped and the stained biofilms were observed with a Leica TCS sp2 CLSM equipped with argon and HeNe lasers.

RNA isolation and microarray hybridization

For RNA isolation experiments, cells were added to 25 mL portions of RPMI-1640 medium in 75-cm2 tissue culture flasks with vented caps. The flasks were incubated statically for 1 h to allow initial adherence of the cells, and then the medium was decanted and replaced with 25 mL portions of fresh RPMI-1640 medium containing 10 μmol/L 10b. We chose this 10b concentration because it still maintained an inhibitory effect on biofilm formation and allowed for recovery of a sufficient cellular mass for RNA extraction. The flasks were then incubated statically for 48 h at 37 °C. A 10b-free control was also included. Total RNA was isolated by the hot phenol method32 and purified with a NecleoSpin® Extract II kit (Machery-Nagel Corp, Germany) following the manufacturer's protocol. A 7925 C albicans genome 70-mer oligonucleotide microarray was obtained from CapitalBio Corporation (Beijing, China). A 1-μg sample of total RNA was used for preparing fluorescent dye-labeled cDNA by the linear mRNA amplification procedure as described33. A DNA–DNA hybridization protocol was used to replace RNA–DNA hybridization in the present study for the sake of reducing cross-hybridization34. The labeled cDNAs were dissolved in 80 μL hybridization solution [3×SSC, 0.2% (w/v) SDS, 5× Denhardt's solution, 25% (v/v) formamide] and denatured at 95 °C for 3 min before hybridization. A sample of the mixed hybridization buffer was placed onto a microarray slide and covered with a glass coverslip. Hybridization was done with a BioMixer™ II (CapitalBio Corp, China). After hybridization, the slides were washed with washing solution 1 (2×SSC, 0.2% SDS) and then with washing solution 2 (2×SSC) at 42 °C for 4 min. Self-hybridization of the control sample was used to evaluate the system noise.

The microarrays were scanned with a LuxScan™ 10KAscanner (CapitalBio Corp, China) at two wavelengths to detect emissions from both Cy3 and Cy5. The image obtained was analyzed with LuxScan™ 3.0 software (CapitalBio Corp, China). Normalization was done on the basis of a Lowess program35. The ratio of Cy5 to Cy3 was calculated for each location on each microarray. To minimize artifacts that arose from low expression values, only genes with raw intensity values>800 counts for both Cy3 and Cy5 were chosen for analysis. A “one class” method for the analysis of microarray software (SAM) was used to identify significantly differentially expressed genes. Genes with a false discovery rate (FDR)<5%, q-value <1% and variation of at least 2-fold in SC5314 biofilms following 10b exposure were identified as significantly differentially expressed genes in two independent experiments. Differentially expressed genes were clustered hierarchically by Gene Cluster 3.0 (Stanford University). DNA sequences were annotated on the basis of the results of BlastN and BlastX searches using the sequencing database of Stanford University (Palo Alto, CA) (http://www-sequence.stanford.edu/group/candida/), GenBank (http://www.ncbi.nlm.nih.gov/BLAST/), and CandidaDB database (http://genolist.pasteur.fr/CandidaDB/).

The entire arrary data have been deposited in the NCBI gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov) and are aceesible through GEO series accession number GSE19226.

Quantitative real-time RT-PCR assay

Real-time RT-PCR was used to confirm microarray results of gene expression changes. An aliquot of the RNA preparations used in the microarray experiments was saved for this follow-up study. Moreover, duplicate independent experiments were conducted to isolate RNA samples according to the protocol described above. First-strand cDNAs were synthesized from 1 μg total RNA in a 20 μL reaction volume using the cDNA synthesis kit for RT-PCR (TaKaRa Biotechnology, Dalian, China) in accordance with the manufacturer's instructions. Real-time PCR reactions were performed with SYBR Green I (TaKaRa), using ABI 7500 Real-Time PCR system (Applied Biosystems Co, California, USA). Gene-specific primers were designed using Discovery Studio Gene software (Accelrys, Inc). The thermal cycling conditions comprised an initial step at 95 °C for 1 min, followed by 40 cycles at 95 °C for 10 s, 55 °C for 20 s, and 72 °C for 30 s. Change in SYBR Green I fluorescence in every cycle was monitored by the system software, and the threshold cycle (CT) was measured. Using 18S rRNA as the internal control, gene expression of SC5314 biofilms treated by 10b relative to that without treatment was calculated using the formula 2−△△CT, where △CT is the CT value of genes of interest minus that of the internal control, and △△CT is the mean △CT value of SC5314 biofilms treated by 10b minus that without treatment. Primer sequences used in real-time RT-PCR assay are listed in Table 1.

Table 1 List of primers used for real-time RT-PCR.
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Statistics

Experiments were performed at least three times. Data are presented as 'mean±standard deviations' and analyzed using the Student's t test where indicated.

Results

In vitro anti-biofilm activity of 10b

10b had been reported to have great antifungal activity36, 37, but its dramatic anti-biofilm activity was unexpected. CLSM showed that normal C albicans biofilm exhibited a typical three-dimensional structure and composed mainly of true hyphae and pseudohyphae (Figure 2A). When cells were preincubated with 10 μmol/L farnesol at 0 h incubation time, biofilm development can not be prevented and still composed mainly of true hyphae and pseudohyphae (Figure 2B). However, after preincubation with 0.1 μmol/L 10b, the typical architecture of biofilms (intertwining mycelial structures and a basal layer of blastospores) was destructed although pseudohyphae and true hyphae were observed (Figure 2C). Moreover, the concentration was increased by 10-fold, the adherent yeast cells were successfully prevented from germination and resulted in scant or nonexistent biofilms (Figure 2D). Effect of 10b on biofilm formation was also assessed by XTT reduction assay as seen in Figure 3. Over 90% inhibition of biofilm formation was observed at 10b concentrations over 15.63 μmol/L. Lower concentrations (7.81 to 15.63 μmol/L) produced about 50% inhibition. Moreover, 10b used at 1.95 to 3.91 μmol/L was still able to reduce about 30%–40% metabolic activity (Figure 3A). Farnesol was also able to inhibit biofilm formation to some extent but the inhibitory effect was relatively poor (Figure 3B). Less than 55% inhibitory efficiency of biofilm formation was observed at farnesol concentrations over 15.63 μmol/L. No significant inhibition was observed at farnesol concentrations lower than 7.81 μmol/L. Furthermore, the SMIC50 value of 10b and farnesol were 7.05 μmol/L and 187.50 μmol/L, respectively, as determined by XTT reduction assay.

Figure 2
figure 2

Morphology alteration of C albicans strain after treated with 10b. Confocal laser scanning microscopy of C albicans biofilms: (A) normal biofilm; (B) cells treated with 10 μmol/L farnesol at 0 h of the incubation time; (C) cells treated with 0.1 μmol/L 10b at 0 h of the incubation time; (D) cells treated with 1.0 μmol/L 10b at 0 h of the incubation time.

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Figure 3
figure 3

Inhibition of biofilm formation by different concentrations of 10b (A) and farnesol (B). biofilm formation (as measured by XTT reduction) is expressed as a percentage of control biofilms. The data are expressed as mean±standard deviation (SD) of three independent experiments. The mean±SD of the mean control values (A492) was 1.083±0.056. Statistically significant differences as determined by Student's t test. bP<0.05, cP<0.01 vs control.

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Next, mature (48 h) biofilms were added with fresh growth medium containing 10b and farnesol, and incubated for another 3 to 24 h. As seen in Table 2, 10b at 10 and 50 μmol/L were able to markedly inhibit the metabolic activity of mature biofilms whenever 10b was added. Moreover, 10b at 50 μmol/L was able to achieve 62.71% inhibitory efficiency after incubation for 24 h. However, only farnesol at 50 μmol/L was able to inhibit metabolic activity of mature biofilms significantly. It was worth mentioning that extension of the incubation time increased the efficiency of 10b and farnesol in inhibiting the metabolic activity of mature biofilms

Table 2 Effects of 10b and farnesol incubated for different times on mature (48 h) biofilms.
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Gene expression responses to 10b exposure

To reveal the mechanism of 10b against biofilms at the molecular level, C albicans cDNA microarrays were used to identify changes in gene expression profiles of C albicans SC5314 biofilms following 10b exposure in two independent experiments. To avoid dye-associated effects on cDNA synthesis, RNA from the 10b-treated group was labeled with Cy5 and Cy3 separately. In each hybridization experiment, duplicate spots were measured as usual. After statistical analysis of six individual comparisons, only the statistically significant (FDR<5%, q-value<1%) genes whose expression level changed by at least 2-fold in all data sets were selected. The data generated from these independent microarray experiments reflected a high level of reliability and reproducibility due to the presented ratios obtained from repeated assays of the same samples and of samples from the independent experiments.

A total of 149 differentially expressed genes were found upon exposure to 10b, of which 69 genes showed a decrease in expression, and 80 genes showed an increase in expression (Table 3 and 4). Ten differentially expressed genes were found to relate to biofilm formation, filamentous or hyphal growth. It was noticed that striking up-regulation of NRG1 and marked down-regulation of ECE1 directly related to the inhibition of biofilm formation. One gene related to specifically hydrolyzing beta-1,3 glucan (XOG1) was significantly increased. Ten down-regulated genes were involved in glycolysis (eg, HXK2), fermentation (eg, ADH1) and active oxygen scavenging (eg, SOD5). Fifteen overexpressed genes were related to the lipid metabolic process. Of them, 13 genes were directly linked to ergosterol biosynthesis, including ERG2, ERG6 and ERG11. Ten genes related to translation were overexpressed following exposure to 10b. Of them, 2 genes involved in negative regulation of transcription were significantly up-regulated.

Table 3 Genes down-regulated in 10b-grown biofilms compared to biofilms without treatment through two independent experiments.
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Table 4 Genes up-regulated in 10b-grown biofilms compared to biofilms without treatment through two independent experiments.
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Validation of microarray data by real-time RT-PCR

To validate the expression of genes identified by microarray analysis, real-time RT-PCR was perfomed in 23 genes of interest selected on the basis of their roles in the specific action mechanism of 10b (eg, ECE1, NRG1, CSA1, ACE2, BMT9, FGR6-10, SLF1, ADH1, HXK2, CDC19, PFK1, GPX2, SOD5, SOD2 and XOG1) and in the responses independent of mechanism of action (eg, SLD1, DAG7, SFL2, GZF3, ERG2, ERG6, ERG11 and ERG24). Total RNA samples from cells treated or untreated with 10b were prepared in parallel for three separate experiments. Real-time RT-PCR reactions were performed in triplicate with independent RNA isolations. It was found that the expression of gene PFK1 was down-regulated as shown by real-time RT-PCR in three independent experiments. In general, there was a good correlation between real-time RT-PCR and microarray data (Figure 4).

Figure 4
figure 4

Gene expression changes in 25 genes of interest in C albicans SC5314 biofilms treated with 10b (10 μmol/L) relative to untreatment: (A) genes related to the specific action mechanism of 10b; (B) genes independent of the action mechanism of 10b. All the genes were examined by real-time RT-PCR with gene-specific primers.

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Effects of ROS on C albicans biofilms

According to Pastuer effect, the tricarboxylic acid cycling process might be enhanced because the expression of genes participating in glycolysis was decreased after exposure to 10b under aerobic circumstances. Besides, the expression of genes encoded superoxide dismutase (SOD) was also decreased. This might result in augmentation of ROS generation (Figure 5). To assess the role of endogenous ROS against C albicans biofilms, XTT reduction assay was performed to investigate the effects of ROS scavenger, ascorbic acid (AA), on anti-biofilm activity of 10b with respect to planktonic cell adhesion, biofilm formation and mature biofilm (48 h) metabolic activity. Table 5 shows that 10b+AA or 10b alone had no effect on planktonic cell adhesion although 10b+AA notably increased metabolic activity of planktonic cells compared to 10b (P<0.01). No significant difference between 10b+AA and 10b was found in inhibitory efficiency of biofilm formation despite the fact that 10b+AA markedly inhibited biofilm formation compared to AA (P<0.01) (Table 6). 10b+AA significantly increased mature biofilm metabolic activity compared to 10b (P<0.05) (Table 6). These results indicated that endogenous ROS augmentation generated by 10b is a pivotal action mechanism to reduce mature biofilm metabolic activity, although elevated endogenous ROS have no impact upon biofilm formation and planktonic cell adhesion. To further verify above conclusion, XTT reduction assay was performed to investigate the effect of a well known ROS inducer MCZ on mature biofilm metabolic activity. As expected, MCZ at 10 μmol/L and 50 μmol/L markedly decreased mature biofilm metabolic activity and the inhibitory efficiency was 39.08% and 58.68%, respectively, after 6 h exposure (Figure 6). Our data further confirmed a direct correlation between endogenous ROS augmentation and inhibition of mature biofilm metabolic activity.

Figure 5
figure 5

Central carbon metabolism in C albicans biofilms during aerobic growth on glucose. The gray ellipses indicate low expression of genes encoded metabolic enzymes participated in aerobic oxidation process after treatment with 10b. The black ellipses indicate augmented tricarboxylic acid cycling process and endogenous reactive oxygen species generation after treatment with 10b. G-6-P, glucose-6-phosphate; F-1,6-2P, fructose-1,6-bisphosphate; G3P, glyceraldehyde-3-phosphate; 1,3-DPGA, 1,3-bisphosphoglycerate; PEP, phosphoenolpyruvate; TCA cycle, tricarboxylic acid cycle.

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Table 5 Effect of 10b combined ascorbic acid (AA) on C albicans planktonic cells adhesion.
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Table 6 Effect of ascorbic acid (AA) on the inhibition of 10b against C albicans biofilm formation and mature biofilm (48 h).
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Figure 6
figure 6

Effect of MCZ incubated for 6 h on mature (48 h) biofilms. MCZ was added to mature (48 h) biofilms and incubation was continued for 6 h at 37 °C. XTT reduction is expressed as a percentage of that of control mature biofilms (48 h) further incubated for 6 h. The results are mean±standard deviations of three independent experiments. Statistically significant differences as determined by Student's t test. bP<0.05, cP<0.01 vs control.

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Discussion

10b is a novel chemical structural antifungal agent with high antifungal activity, a broad antifungal spectrum and potentially low toxicity36, 37. In this study, we found that 10b had strong anti-biofilm activity. To further investigate the action mechanism, cDNA microarray study was performed in C albicans SC5314 biofilms treated or untreated with 10b. The results of real-time RT-PCR showed that differentially expressed genes were involved in multiple biochemical functions. Our particular interest was the striking changes of biofilm formation related genes and energy-metabolism-related genes: glycolysis-related genes, fermentation-related genes and active oxygen scavenging-related genes.

It was found in this study that biofilm formation was directly related to striking down-regulation of ECE1 and marked up-regulation of NRG1. ECE1, a hypha-specific gene, is involved in the process of cell extension, the first morphological sign of which is the emergence of a germ tube approximately 90 min after induction38. Reports have confirmed that ECE1 promoter is strongly repressed by Nrg1p, a sequence-specific DNA-binding protein, under non-inducing conditions39, 40. Nrg1p, endoed by NRG1, plays important roles in the regulation of yeast-hyphal morphogenesis, and represses a subset of Tup1-regulated genes, including known hypha-specific genes and other virulence factors. Most of these genes contain an Nrg1p response element (NRE) in their promoter. Nrg1p interacts specifically with an NRE in vitro39. Inactivation of Nrg1p in C albicans causes flamentous and invasive growth, derepresses hypha-specific genes and increases sensitivity to some stresses39, 40, 41. Our data indicate that striking down-regulation of ECE1 and marked up-regulation of NRG1 induced by 10b might play a pivotal role during biofilm formation.

Another interesting result was the change of energy-metabolism-related genes after 10b treatment: global down-regulation of glycolysis-related genes, fermentation-related genes and antioxidant defense genes. According to Pasteur effect that aerobic oxidation could inhibit glycolysis (alcohol-facient fermentation) under aerobic circumstances, the tricarboxylic acid cycling process might be enhanced following exposure to 10b because expression of the genes participating in glycolysis especially encoding two rate-limiting enzymes genes, HXK2 and PFK1, was markedly decreased. Besides, expression of the gene encoded superoxide dismutase (SOD5) was also decreased. Together, endogenous ROS generation might be markedly augmented and experiment verified our deduce (data not shown). To determine whether endogenous ROS augmentation was the major mechanism of 10b against C albicans biofilms, we performed XTT reduction assay to investigate the effect of anti-oxidant AA on the anti-biofilm activity of 10b and the effect of a well known ROS inducer MCZ on mature biofilm metabolic activity. Taken together, our results suggest that the mechanism of 10b against mature biofilms is associated with the augmentation of endogenous ROS by affecting aerobic respiration in mitochondria.

Endogenous ROS, a natural byproduct of normal cellular metabolism , derives from mitochondrial respiratory chain electron leakage and plays important roles in cellular components damage and DNA cell signaling42, 43. Reports have confirmed that increase of intracellular ROS production is involved in the mechanism of several antifungal agents44, 45. Furthermore, it was found in our previous study that decreased endogenous ROS generation contributes to drug resistance of C albicans46. However, the mechanisms by which Candida biofilms resist the action of antifungal agents are not known. In our present study, we found that the augmentation of endogenous ROS generated by 10b significantly reduced mature biofilm metabolic activity, and when the augmentation endogenous ROS is scavenged after addition of anti-oxidant AA, mature biofilm metabolic activity increased markedly. Our data indicate that ROS augmentation might be a major mechanism of 10b against C albicans mature biofilms. This was confirmed by a well known ROS inducer MCZ, which was able to markedly reduce mature biofilm metabolic activity at 10 μmol/L.

Drug binding assays and cell wall component modification studies suggest that cell wall changes may contribute to antifungal biofilm drug resistance13. Beta-1,3-glucan is the major structural polysaccharide of the fungal cell wall and is thought to be the main component of the three-dimensional matrix surrounding biofilm cells and closely tied to biofilm formation47. In our study, we found that expression of gene XOG1, encoding exo-1,3-beta-glucanase enzyme, increased dramatically. This indicated that up-regulated XOG1 might be another pathway to inhibit biofilm formation because exo-1,3-beta-glucanase enzyme specifically hydrolyzes beta-1,3 glucan, resulting in destruction of the matrix of C albicans biofilms and cell wall48.

In conclusion, the results of the present study show that 10b treatment altered the expression of biofilm formation-related genes, impaired the ability of C albicans to change its morphology, decreased beta-1,3 glucan levels in the biofilm matrix and cell wall, and ultimately blocked C albicans to form mature biofilms. In addition, mitochondrial aerobic respiration shift and endogenous ROS augmentation also contributed to reducing mature biofilm metabolic activity. This might provide useful information for the development of new strategies to reduce the incidence of device-associated infections.

Author contribution

Yuan-ying JIANG and Yong-bing CAO designed research; Rong-mei LIANG performed the research and wrote the paper; You-jun ZHOU and Hui TANG synthesized 10b; Yi XU, Ping-hui GAO, Bao-di DAI and Feng YANG critically read the manuscript.